Chromatographic purification of adeno-associated virus (AAV)

ABSTRACT

PCT No. PCT/US96/13872 Sec. 371 Date Jun. 29, 1998 Sec. 102(e) Date Jun. 29, 1998 PCT Filed Aug. 30, 1996 PCT Pub. No. WO97/08298 PCT Pub. Date Mar. 6, 1997The present invention relates to the purification of large scale quantities of active (infectious) adenovirus and AAV, especially for use in therapeutic applications. In particular, the invention provides improved methods for contacting such viruses with suitable chromatographic materials in a fashion such that any damage to the virus, particularly to surface components thereof, resulting from contact with such chromatographic materials is minimized or eliminated. The result is the ability to rapidly and efficiently purify commercial level quantities of active (infectious) virus suitable for use in therapeutic applications, e.g. gene transfer/therapy procedures.

The present application is a national stage application filed under 35USC chapter 371, from the international application No. PCT/US96/13872,filed on Aug. 30, 1996, which was a continuation in part of U.S.provisional patent application Ser. No. 60/002,967, filed on Aug. 30,1995, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the purification of large scalequantities of active (infectious) adenovirus and AAV, especially for usein therapeutic applications. In particular, the invention providesimproved methods for contacting such viruses with suitablechromatographic materials in a fashion such that any damage to thevirus, particularly to surface components thereof, resulting fromcontact with such chromatographic materials is minimized or eliminated.The result is the ability to rapidly and efficiently purify commerciallevel quantities of active (infectious) virus suitable for use intherapeutic applications, e.g. gene transfer/therapy procedures.

BACKGROUND OF THE INVENTION

Molecular therapy of disease often involves the administration ofnucleic acid to the cells of interest in order to confer a therapeuticbenefit. Most commonly, recombinant viruses are engineered which takeadvantage of the natural infectivity of viruses and their ability totransport heterologous nucleic acid (transgene) to a cell. Widespreaduse of such recombinant viral vectors depends on strategies for thedesign and production of such viruses.

Most attempts to use viral vectors for gene therapy have relied onretrovirus vectors, chiefly because of their ability to integrate intothe cellular genome. However, the disadvantages of retroviral vectorsare becoming increasingly clear, including their tropism for dividingcells only, the possibility of insertional mutagenesis upon integrationinto the cell genome, decreased expression of the transgene over time,rapid inactivation by serum complement, and the possibility ofgeneration of replication-competent retroviruses (Jolly, D., Cancer GeneTherapy 1:51-64, 1994; Hodgson, C. P., Bio Technology 13:222-225, 1995).

Adenovirus is a nuclear DNA virus with a genome of about 36 kb, whichhas been well-characterized through studies in classical genetics andmolecular biology (Horwitz, M. S., "Adenoviridae and Their Replication,"in Virology, 2nd edition, Fields, B. N., et al., eds., Raven Press, NewYork, 1990). Adenovirus-based vectors offer several unique advantagesfor delivering a therapeutic transgene to a cell, including, inter alia,tropism for both dividing and non-dividing cells, minimal pathogenicpotential, ability to replicate to high titer for preparation of vectorstocks, and the potential to carry large inserts (Berkner, K. L., Curr.Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy1:51-64, 1994).

Adeno-associated virus (AAV) is a single-stranded non-pathogenic DNAvirus which is capable of integrating into the genome of an infectedcell. This feature of the virus life cycle has focused attention on theuse of AAV as a gene therapy vehicle (creating a recombinantadeno-associated vector, rAAV) to deliver a gene of interest for genetherapy. The ability of AAV to insert a therapeutic gene into the cellgenome facilitates persistent expression of the gene of interest andreduces the need for repeated dosing of a gene therapy vector.

Current methods for the purification of adenovirus and adeno-associatedvirus (AAV) involve the use of density gradient centrifugation, whichdoes not easily allow for large scale production of virus stocks fortherapeutic use. A further limitation to widespread use of AAV vectorsis the general lack of any adequate purification methods which yieldhigh titers of AAV, while removing contaminating adenovirus required forthe propagation of AAV vector stocks.

Ion-exchange, affinity chromatography and gel filtration are widely usedcolumn chromatography tools in protein purification. Until recently,however, these methods have been inapplicable to purification ofadenoviruses. Such techniques have resulted in damage to the viruses,thereby reducing their ability to bind and infect a target cell.Provisional U.S. patent application Serial No. 60/002,967, filed Aug.30, 1995, set forth parameters for purifying infectious adenovirusutilizing chromatographic fractionation techniques as described morefully herein. Recent studies have shown that column chromatography maybe used in the purification of recombinant adenovirus (Huyghe et al.,Human Gene Therapy 6:1403-1416, 1995).

Column chromatography, using other systems such as so-called"macroporous" resins, which comprise beads having pores therein, theaverage diameter of which is approximately the same as the diameter ofadenovirus (diameter=about 80 nm, excluding the fibres and about 140 nmwith the fibre molecules), have not resulted in the recovery ofinfectious adenovirus. The most likely reason for this is that thepassage of adenovirus through such resins shears the fibres from theviral surface through intimate contact of the virus with the pores inthe beads. The adenovirus fibre molecules, inter alia, are believed tobe involved in the virus ability to bind to and infect target cells.Thus, damage or loss of the fibre molecules (as well as other surfacemolecules) by such prior art column methods results in the recovery ofinactive (non-infectious) virus.

As is well known in the art, AAV propagation requires the use of helpervirus, such as adenovirus. The requirement for helper virus complicatespurification of AAV. Current approaches to AAV purification involvelysing of AAV infected cells using repeated cycles of freeze-thawingfollowed by the use of density gradient centrifugation to fractionatethe cell lysate in order to obtain infectious AAV, free of cellularcontaminants and substantially free of helper virus (such as adenovirus)required for AAV propagation. (Flotte et al., Gene Therapy 2:29-37,1995; Chiorini et al., Human Gene Therapy 6:1531-1541, 1995; Fisher etal., J. Virol. 70: 520-532, 1996). Standard purification techniquesgenerally result in very low yields (0.3-5%) of active (infectious)virus. Moreover, because of the helper-virus requirement, it has beendifficult to obtain AAV that is totally free of the helper (e.g.adenovirus).

SUMMARY OF THE INVENTION

The present invention is directed to methods for the purification ofcommercially useful quantities of infectious adenovirus and AAV,especially for therapeutic use.

The present invention avoids the problems associated with prior artmethods of purifying infectious virus and relies on chromatographicfractionation techniques which provide for large scale separation ofinfectious adenovirus that are useful in gene transfer of therapeutictransgenes to a host. Thus, the present invention provides improvedmethods for contacting therapeutically-useful viruses with suitablematerials used in chromatographic fractionation techniques in a fashionand under conditions such that the viruses, especially surfacecomponents thereof believed required for infectivity, are not damaged bysuch contact.

Especially in regard to adenovirus purification, the inventionencompasses several design considerations involved in these improvedmethodologies. These design considerations are related in that theyaccomplish a similar objective--minimizing or eliminating damage to thevirus by contact with various chromatographic materials used inpurification. In particular, the approaches are intended to obviate theeffect of the openings or pores in such materials which are involved inthe partitioning of biological molecules.

In one aspect of the invention, a "batch"-type technique may be used. Inthis aspect, the virus is mixed with a suitable chromatographic materialrather than subjected to a "flow-through" procedure. It is believed thatwith this approach, the virus particles are less likely to enter thepores in the beads and suffer damage.

A second aspect of the invention involves using chromatographicmaterials in which the pore size of the support material is so smallthat the virus cannot enter the pores during chromatographicfractionation (e.g. in a column or membrane). The reduction in pore sizeof such chromatographic materials can be accomplished, e.g. by increasedcross-linking of the support matrix. The reduction in pore size preventsthe viruses from entering the openings in the beads where they can bedamaged.

Alternatively, chromatographic materials may be used which containstructures, e.g. "tentacles," that prevent viruses from getting close tothe pores in the matrix material. Again, this serves to prevent orminimize damage to the virus particles.

In a third aspect of the invention, chromatographic materials may beused wherein the matrix of the materials contains openings or pores thatare very large in size, i.e. pores that have a diameter significantlylarger than the diameter of the adenovirus particles. Thus, the viruscan be partitioned through the pores in such chromatographic materialswithout being damaged.

Based on these design considerations, the purification methods of thepresent invention allow for the use of wide variety of commerciallyavailable chromatographic materials known to be useful in fractionatingbiological materials. Useful support matrices of such materials caninclude, inter alia, polymeric substances such as cellulose or silicagel type resins or membranes or cross-linked polysaccharides (e.g.agarose) or other resins. Also, the chromatographic materials canfurther comprise various functional or active groups attached to thematrices that are useful in separating biological molecules.

As such, the methods of the invention also exploit the use of affinitygroups bound to the support matrices with which the viruses interact viavarious non-covalent mechanisms, and can subsequently be removedtherefrom. Preferred approaches presented in the present inventionexploit ion-exchange (especially anion-exchange) type interactionsuseful in chromatographic fractionation techniques. Other specificaffinity groups can involve, inter alia, the use of heparin andvirus-specific antibodies bound to the support matrix. Of considerationin the choice of affinity groups in virus purification via the presenttechniques is the avidity with which the virus interacts with the chosenaffinity group and ease of its removal without damaging viral surfacemolecules involved in infectivity.

Commercial-scale production of relatively high molecular weight virusspecies (e.g. adenovirus and AAV) at high yields of active (infectious)virus is achieved with these methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic diagram of Acti-Mod® Cartridge resin.

FIG. 2: Chromatogram showing elution profile of adenovirus fromDEAE-MemSep® resin. Elution peak for adenovirus is labelled.

FIG. 3: Chromatogram showing elution profile of adenovirus fromSuperdex® 200 resin. Elution peak for adenovirus is labelled.

FIG. 4: SDS-PAGE analysis of adenovirus purified by DEAE and gelfiltration chromatography (Lane A), compared to CsCl gradient purifiedvirus (Lane B).

FIG. 5: SDS-PAGE analysis of adenovirus purified by heparin, DEAE andSuperdex® chromatography (Lane B), compared to CsCl gradient purifiedvirus (Lane A).

FIG. 6: Densitometric analysis of adenovirus purified by heparin, DEAEand Superdex® chromatography (A), compared to CsCl gradient purifiedvirus (B).

FIG. 7: Chromatogram showing elution profiles of AAV from (A) Superdex®200 resin and (B) DEAE-MemSep® resin.

FIG. 8: SDS-PAGE analysis of two fractions of AAV purified by Superdex®and DEAE chromatography (Lanes A and B).

FIG. 9A: Ceramic hydroxyapatite chromatography of an AAV/adenoviruscontaining 293 cell lysate. Elution peaks for both AAV and adenovirusare labelled.

FIG. 9B: DEAE-MemSep® chromatography of an hydroxyapatite AAV-containingeluate. Elution peaks for both AAV and adenovirus are labelled.

FIG. 9C: Cellufine® sulfate Chromatography of an AAV-containing DEAEeluate. The eluted AAV peak from the resin is labelled.

FIG. 10: Coomassie-stained SDS-PAGE analysis of the AAV containingfractions recovered from various columns: lane 1: hydroxyapatite load;lane 2: hydroxyapatite eluate; lane 3: DEAE eluate; lane 4: Cellufine®sulfate eluate; and lane 5: Cellufine® sulfate eluate (100 μl). Controlprotein standards (in kD) are shown in the left column.

FIG. 11: SDS-PAGE analysis of AAV purified by ceramic hydroxyapatite,DEAE and Cellufine® sulfate chromatography.

FIG. 12: Coumassie-stained protein gel of AAV purified by ceramichydroxyapatite, DEAE and Cellufine® sulfate chromatography (Lane 1),compared to CsCl gradient purified AAV (Lanes 2-4).

FIG. 13: Immunoblot of AAV purified by ceramic hydroxyapatite, DEAE andCellufine® sulfate chromatography (Lane AAV), using an anti-Repantibody, compared to Rep controls.

FIG. 14: Schematic diagram of pTRlacZ.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to chromatographic fractionationmethods adaptable for large scale for the purification of active(infectious) adenovirus and adeno-associated virus (AAV), especially foruse in therapeutic applications, such as gene transfer, including genetherapy. The chromatography methods of the invention are intended toreplace the current non-scaleable method of density-gradientultracentrifugation of virus purification and allow for production ofactive (infectious) viruses on an industrial scale. The designstrategies for purification of the two viruses are interrelated. Asafore-mentioned, propagation of AAV requires the presence ofhelper-virus components, most commonly provided by adenovirus. Thepurification of AAV has been problematic because of adenoviralcontamination.

The chromatographic fractionation techniques of the invention offerseveral advantages over prior virus purification procedures based solelyupon centrifugation: the methods are rapid; the protocols are efficient,permitting separation of milligram quantities of virus in a single run;virus integrity is not compromised; and high yields of infectious virusare obtained.

Purification of Adenovirus

As aforementioned, prior attempts to adapt conventional chromatographicprocedures and materials, such as those routinely used in thepurification of proteins or other viruses, to the purification ofadenovirus, have been unsuccessful. It is believed that in applying suchprocedures, insufficient care has been taken to protect the adenovirusfrom damage, and in particular, to macromolecular components thereofnecessary for infectivity. Without being limited as to theory, it isrecognized that particular adenoviral proteins, including mostparticularly the protein species known as "fibre," are involved in thebinding of adenovirus to target cells during infection. Althoughnumerous copies of such proteins may be found on each adenovirus, viralinfectivity is relatively low for most cell types, and thus damage toeven a small portion of, for example, the fiber molecules or other viralmacromolecules can substantially prevent the establishment of successfulinfection. Maintaining high infectivity is therefore of considerableimportance with respect to the commercial scale production of adenoviralvectors intended for therapeutic use, such as for gene therapy.

Accordingly, there are provided a wide variety of chromatography methodsto take properly into account the fragile nature of adenovirusparticles. As presently disclosed, a wide variety of conventionalchromatographic materials including matrix or support materials, and theactive (binding) groups routinely coupled thereto, are useful in thepractice of the invention.

The methods described here in permit retrieval of purified adenoviralparticles at high concentration in aqueous media without damage toadenoviral components. Similarly, the methods are suited to thepreparation of milligram quantities of virus.

Adenovirus is isolated from virus-infected cells, for example, 293cells. Cells may be infected at high multiplicity of infection (MOI) inorder to optimize yield. Any method suitable for recovering virus frominfected cells may be utilized. Preferred techniques for the recovery ofvirus from infected cells include freeze-thawing and the use of amicrofluidiser. However, in order to make purification of viruses ascaleable process it is preferable to use procedures which lyse thevirus infected cells without repeated freeze thawing and to removecellular debris from the cell;lysate without centrifugation. Optimalconditions for lysis of virus infected 293 cells for release of activevirus may be achieved using a pressure cell, e.g. a Microfluidiserpressure cell (Microfluidics, Newton, Mass.). Ultrafiltration of thelysate using, for example, a Minitan system (Millipore, Bedford, Mass.),which comprises a High Resolution Tangential Flow System, can be used tofurther concentrate the virus fraction prior to chromatographic fractiontechniques.

The adenovirus-containing lysate so obtained may then be subjected tothe chromatographic fractionation techniques of the invention. In regardto adenovirus purification, the invention encompasses several designconsiderations involved in these improved methodologies. These designconsiderations are related in that they accomplish a similarobjective--minimizing or eliminating damage to the virus by contact withvarious chromatographic materials used in purification. In particular,the approaches are intended to obviate the effect of the openings orpores in such materials which are involved in the partitioning ofbiological molecules.

In one aspect of the invention, a "batch"-type technique may be used. Inthis aspect, the virus is mixed with a suitable chromatographic materialrather than subjected to a "flow-through" procedure. It is believed thatwith this approach, the virus particles are less likely to enter thepores in the beads where they can become damaged.

A second aspect of the invention involves using chromatographicmaterials in which the pore size of the support material is smaller thanthat of the virus particles so that the virus cannot enter the poresduring chromatographic fractionation (e.g., in a column or membrane).The reduction in pore size of such chromatographic materials can beaccomplished, e.g. by increased cross-linking of the support matrix. Thereduction in pore size prevents the viruses from entering the openingsin the beads where they can be damaged.

Alternatively, chromatographic materials may be used which containstructures, e.g. "tentacles," that prevent viruses from getting close tothe pores in the matrix material. Again, this serves to prevent orminimize damage to the virus particles.

In a third aspect of the invention, chromatographic materials may beused wherein the matrix of the materials contains openings or pores thatare very large in size, i.e., pores that have a diameter significantlylarger than the diameter of the adenovirus particles. Thus, the viruscan be partitioned through the pores in such chromatographic materialswithout being damaged.

Based on these design considerations, the purification methods of thepresent invention allow for the use of wide variety of commerciallyavailable chromatographic materials known to be useful in fractionatingbiological materials. Useful support matrices of such materials caninclude, inter alia, polymeric substances such as cellulose or silicagel type resins or membranes or cross-linked polysaccharides (e.g.agarose) or other resins. Also, the chromatographic materials canfurther comprise various functional or active groups attached to thematrices that are useful in separating biological molecules.

As such, the methods of the invention also exploit the use of affinitygroups bound to the support matrices with which the viruses interact viavarious non-covalent mechanisms, and can subsequently be removedtherefrom. Preferred approaches presented in the present inventionexploit ion-exchange (especially anion-exchange) type interactionsuseful in chromatographic fractionation techniques. Other specificaffinity groups can involve, inter alia, the use of heparin andvirus-specific antibodies bound to the support matrix. Of considerationin the choice of affinity groups in virus purification via the presenttechniques is the avidity with which the virus interacts with the chosenaffinity group and ease of its removal without damaging viral surfacemolecules involved in infectivity.

The following types of chromatographic materials are suitable for thebatch-type chromatographic methods discussed above.

The teachings of the present disclosure also make possible the use ofother commercially available chromatographic materials, if only in batchform. Although not a preferred embodiment of the invention, once it isunderstood that adenovirus can be damaged by contacting suchchromatographic materials, purification procedures can be redesigned tominimize damage to the virus. To the extent that many polymer materialscontain pores which can damage any contacting adenovirus, such damagecan be limited by minimizing, for example, column pressure, therebylimiting entry of adenovirus into matrix pores. In the simplest examplethereof, the purification step is simply conducted in batch form.Polymer materials useful according to this aspect of the inventioninclude the products Heparin Sepharose High Performance, of Pharmacia;macroporous hydroxyapatite such as Macro-Prep Ceramic Hydroxyapatite,Bio-Rad, Richmond, Calif.; and cellufine sulfate from Amicon.

Chromatography materials comprising polymers having sufficient matrixcrosslinking such that interaction of adenovirus with any pore spacesthereof is minimized, wherein also are present any of a number ofbinding groups (including, for example, ion exchange groups or heparins)having affinity for the adenovirus. Representative heparinized polymersuseful in the practice of the invention include those having about 6% ormore of crosslinking, such as Heparin Superflow Plus® (or Sterogene), a6% crosslinked heparin agarose. Such an agarose matrix has an exclusionlimit of about 6 million daltons which is expected to be much lower thanthat of a 4% crosslinked product, such as Heparin Agarose (SigmaChemical Co.). In one experiment, recovery of adenovrius in infectiveform was substantially improved when the 6% rather than the 4% productwas used. Additional polymers containing heparin groups that are usefulin the practice of the invention include Heparin Sepharose® CL6B(Pharmacia).

Once the advantages of carefully controlling crosslinking areunderstood, it is apparent to those skilled in the art could substituteany number of polymer materials composed of any number of recognizedmatrix materials and binding groups such that the adenovirus is notdamaged by contact therewith.

As to the second design consideration provided, representative ofchromatographic materials containing functional groups that interacteffectively with adenovirus but which have a design that minimizesaccess of the virus to any pore spaces thereof (such as about 0.1micron), which can damage the virus, and in particular, the fiberprotein thereof, are the following.

Likewise, so-called tentacled polymers containing core regions to whichare attached polymerized chains of varying lengths, and to which may beattached further functional groups; for example, Fractogel Tentacle IonExchange Media available from E. Merck, Wakefield, R.I. thesechromatographic materials are described as having an insoluble matrixcopolymerized from oliogethyleenglycol, glycidylmethacrylate, andpenta-erythrold-imethacrylate, to which are grafted polymerized chainsof acrylamide derivatives, ending in ion exchanging groups such as DEAE,or quaternary aminoethyl, quaternary ammonimum, DMAE, TMAE, and thelike, or example, SO₃ or carboxyl. It is within the practice of theinvention to utilize similar "tentacled" materials.

Preferred, chromatographic materials regarding the third design comprisematrixes having pores of at least about 0.1μ (the sign of anadenovirus), but preferably at least about 1μ.

Such materials can be cellulose membrane or silica membrane cartridgescharacterized by high substrate specificity for target promoters,negligible binding of non-specific proteins, and a pore size (˜1.2 μm)which is sufficient for purification of the largest known spheroidalviruses. The cartridge design, which consists of a stack of low-bindingcellulose or silica membrane filters, is suited to high flow rates,while the large pore size (1.2 μm) of the cartridges eliminates thediffusion associated with beaded gel resins packed in columns.

For example, the ACTI-MOD® (American International, Natick, Mass.)cartridges consist, for example, of sheets of microporous silica/PVC.These silica sheets have a large surface area with numerous uniformpores (See FIG. 1, panel A). The pores are lined with silica to whichdifferent active side chains may be attached, e.g. DEAE or heparinstructural units. During chromatography, the movement of virus throughthe highly porous silica/PVC sheets of ACTI-MOD® (or through theopenings in similarly effective MemSep® (Millipore, Bedford, Mass.)cartridges) permits direct contact of the virions with the activatedsilica surface (FIG. 1, panel B). Such contact permits appropriatepartitioning while, at the same time, avoiding adverse interaction ofthe virions with pores in the beads of the resin that are approximatelythe same size, or slightly smaller, than the size of the encapsulatedvirions themselves that occurs with the use of traditional beadedgel-type resins. Such adverse interaction may involve damage to virionsurface components, including for example fibre protein, therebyreducing the ability of the virion to successfully infect target cells.

Additional polymer products which are useful in the practice of theinvention are those which also are large enough not to damage the virionand include, inter alia, spiral preparative chromatography modules, suchas the CycloSep™ module (American International Chemical Inc., Natick,Mass.). The CycloSep™ spiral purification column has a matrix comprisinga microporous plastic sheet (MPS®), with an integral rib designavailable in various spacing configurations. The matrix is wound into aspiral and is coated with silica. The resultant hydrophilic surface canbe derivatized with various affinity ligands such as heparin, or thoseused in ion-exchange chromatography, such as DEAE and CM.

Anion-exchange chromatography may be performed utilizing variousfunctional moieties known in the art for anion-exchange techniques,including, but not limited to, DEAE, (diethyl aminoethyl), QAE(quaternary aminoethyl), and Q (quaternary ammonium). These functionalmoieties may be attached to any suitable resin useful in the presentinvention, including the cellulose and silica resins described herein.For example, DEAE may be attached to various resins, including celluloseresins, in columns such as DEAE-MemSep® (Millipore, Bedford, Mass.)Sartobind™ membrane absorbers (Sartorius, Edgewood, N.J.) and silicaresins such as ACTI-MOD®, (American International Chemical, Natick,Mass.).

Cation-exchange chromatography also may be used for adenoviruspurification, including, but not limited to, the use of such columns asSP MemSep® (Millipore, Bedford, Mass.), CM MemSep® (Millipore, Bedford,Mass.), Fractogel® SO₃ -(EM Separation Technology, Gibbstown, N.J.) andMacroprep S™ (BioRad, Melville, N.Y.), as well as heparin-based resins.Heparin ACTI-MOD® Cartridge (American International Chemical Inc.,Natick, Mass.), and POROS® Perfusion chromatography media (BoehringerMannheim) represent additional examples of this embodiment of theinvention.

Other affinity ligands which may be used to purify adenovirus includeanti-adenovirus antibodies attached to suitable resins as providedherein as well as others known to those skilled in the art.

Preferably, purification of adenovirus includes the following steps:pressure lysis of 293 cells infected with adenovirus in the presence ofa detergent such as Tween-80 and recovery of virus in the lysate;clarification of lysate by passage through a 3 μm glass fiber filter anda 0.8 μm cellulose acetate filter; heparin chromatography using anACTI-MOD® silica cartridge where adenovirus is recovered in theflow-through fraction; and DEAE-ion exchange chromatography using aMemSep® cartridge. Bound adenovirus is eluted from the resin using500-700 mM NaCl in a suitable buffer; and gel filtration (sizeexclusion) chromatography of the DEAE-eluate, where adenovirus isrecovered in the void volume of the column eluate.

Gel filtration chromatography uses, for example, resins such asSuperdex® (Pharmacia, Piscataway, N.J.) to purify can be used to recoveractive adenovirus away from any contamination process. Such resins havea very small pore size (exclusion limit 2 million daltons) which resultsin the adenovirus being completely excluded from the beads of the resinand eluting in the void volume of the column. The partitioning functionsin a similar fashion to a protein de-salting column. START Adenoviruspurification may be determined by assaying for viral proteins, using,for example, Western blotting or SDS-PAGE analysis. The identificationof adenoviral DNA may be used as an indicator of virus recovery, using,for example, slot blot analysis, Southern blotting, or restrictionenzyme analysis of viral DNA. Purification is evidenced by thepredominance of viral proteins or nucleic acid in an assayed sample.

The identification of adenovirus particles may also be used to assayvirus purification, using, for example, the spectrophotometricabsorbance at 260 nm of a purified fraction, or the observance of virusparticle by, for example, electron microscopy.

The recovery of infectious adenovirus may be determined by infection ofa suitable host cell line (e.g., 193 cells) with a chromatographedsample. Infectious adenovirus may be identified and titrated by plaqueassays. Alternatively, infected cells may be stained for the abundantadenoviral hexon protein. Such staining may be performed by fixing thecells with acetone:methanol seven days after infection, and stainingwith a polyclonal FITC-labeled anti-hexon antibody (Chemicon, Temecula,Calif.). The activity of a purified fraction may be determined by thecomparison of infectivity before and after chromatography.

The use of other columns in the methods of the invention is within thescope of the invention directed to purification of adenovirus.

Since, as discussed above, AAV propagation requires helper adenovirus,which can purify with and contaminate AAV preparations, AAV purificationtakes advantage of chromatographic materials which damage adenovirus(i.e.g, through contact with the pores of such materials) in thepurification of AAV. Thus, AAV purification uses chromatographicmaterials that are not preferred for adenovirus purification. Forexample, such materials can include the so-called "macroporous" resinsdiscussed above, whose pore size is approximately that of adenovirus. Ingeneral, it is advantageous to select matrices with pore sizes thatwould damage adenovirus leading to its inactivation during purification.

Purification of AAV

The initial lysis of AAV-infected cells can be accomplished via methodsthat chemically or enzymatically treat rather than physically manipulatethe cells in order to release infectious virions. Such methods includethe use of nucleases (such as benzonase®; DNAse) to enzymaticallydegrade host cell, non-encapsulated or incomplete adenoviral DNA.Proteases, such as trypsin, can be used to enzymatically degrade hostcell,adenoviral or free AAV proteins. Detergents, surfactants and otherchemical agents known in the art can also be used alone or inconjunction with enzymatic treatment.

AAV-infected cells can be further lysed by application to a pressurecell, such as a Microfluidiser pressure cell, wherein the intensifiedpumping system employs an accelerated suction stroke and a long slowpressure stroke to create a pressure profile of briefly interruptedconstant pressure. This pressure is then used to lyse the AAV-infectedcells, while gently retaining maximal activity of the AAV. A furtheradvantage of the use of such pressure techniques for lysis is that suchmethods can be applied to scaled up cell culture conditions, includingpropagating cells on microcarriers. Alternatively, a French PressureCell (Baxter, Deerfield, Ill.), Manton Goulin Homgeniser (Baxter,Deerfield, Ill.) or Dynomill can be used. The resulting lysate can thenbe clarified by filtration through glass fiber filters or celluloseacetate filters. Alternatives include Millex® Durapore (Millipore,Bedford, Mass.) and Gelman Science Tuffryn® filters (Gelman Science, AnnArbor, Mich.). Filter sizes which may be used include 0.8 μm and 0.45μm. If vacuum filtration is not used, glass wool also may be employed toclarify the lysate.

The present invention also relates to methods for the separation of AAVfrom adenoviral and cellular proteins in the cell lysate, using columnchromatography. The advantage of column chromatography over currentnon-scaleable methods of density-gradient ultracentrifugation is thatlarge quantities of AAV can be produced. Another advantage of usingcolumn chromatography for the purification of AAV is that it effectivelyremoves contaminating adenovirus which is used routinely as a helpervirus in the production of AAV.

Suitable column chromatography methods to fractionate infected celllysates for large-scale purification of AAV include the use of sulfatedresins, such as Sterogene-S (Sulfated Hi Flow) (Sterogene, Carlsbad,Calif.), Spherilose-S (Isco, Lincoln, Nebr.), Cellufine® sulfate(Amicon, Beverly, Mass.). Such sulfated resins are capable of removingcontaminating adenovirus from AAV using elution buffers containing about400-500 mM, preferably about 475 mM NaCl.

DEAE containing resins used in AAV purification, include, but are notlimited to, Puresyn DEAE (Puresyn, Malvern, Pa.), EM Merck Tentacle DEAE(Merk, Whitehouse Station, N.J.), Sterogene Superflow Plus DEAE(Sterogene, Carlsbad, Calif.), macroporous DEAE resins (Biorad,Melville, N.Y.), DEAE ACTI-MOD® (American International, Natick, Mass.),DEAE MemSep® (Millipore, Bedford, Mass.), all of which are capable ofremoving contaminating adenovirus.

Em Merck Tentacle DEAE is an ion exchange media consisting of a matrixcopolymerized from oligoethyleneglycol, glycidymethacrylate andpentaerythroid to which are grafted polymerized chains of acrylamidederivatives approximately 15-50 units in length. Sterogene SuperflowPlus® DEAE consists of a 6% cross-linked agarose to which is attachedDEAE reactive groups. Macroporous DEAE resins are rigid macroporoushydrophilic supports with pore sizes of 80-100 nm, where the DEAEreactive groups are attached to the hydrophilic support. DEAE Acti-Mod®cartridge consists of sheets of microporous silica/PVC. These silicasheets have a large surface area with numerous uniform pores. The poresare lined with silica to which may be attached active side chains suchas DEAE. This type of macroporous structure has pores of about 12,000 Å,or 1.2 μm, in width. In the DEAE MemSep® resin, the DEAE groups arecovalently linked to the polymer matrix cellulose. A suitable elutionbuffer for the recovery of AAV from such resins comprises 200 mM NaCl ina phosphate buffer, pH 7.5.

Purification of AAV also utilizes hydroxyapatite resins, including theceramic hydroxyapatite resins from Biorad (Melville, N.Y.). The recoveryof AAV from such hydroxyapatite resins utilizes elution with a 100-135mM phosphate buffer (pH 6.4, 10-400 mM phosphate gradient. The column isfirst washed with 30 mM phosphate and AAV elutes around 135 mMphosphate).

In a particular aspect of the invention, chromatography on cellulose orsilica membrane resins is employed in conjunction with the use ofmacroporous resins in for effective large-scale purification of AAV.Examples of macroporous resins include the BioRad macroporous series(Melville, N.Y.) or the DEAE-Thruput (6% cross-linked agarose)(Sterogene, Carlsbad, Calif.). Silica or cellulose membrane resinsinclude the DEAE-MemSep™ 1010 HP (Millipore), the ACTI-MOD® cartridge,or the CycloSep™ (American Chemical International) spiral purificationcolumn. Previous studies with macroporous resins showed them not to bevery useful for the purification of adenovirus because the 80 nm poresize of the beads excludes and damages adenovirus particles having adiameter of 140 nm. However, this size limitation is an advantage forAAV purification, because these resins can remove contaminatingadenovirus from AAV preparations based on the different sizes of thevirion particles. It is well within the skill of those in the art toselect macroporous resins for their ability to separate AAV fromadenovirus based on discriminating pore size of the resins.

In a particular embodiment of the invention, pressure lysis ofAAV-infected cells in the presence of detergent yields a cell lysatewhich is clarified by filtration. The lysate is then applied to a seriesof columns in order to separate AAV from cellular proteins andcontaminating adenovirus. A preferred series of column separationsincludes the use of ceramic hydroxyapatite, DEAE ion-exchange,Cellufine® sulfate, and zinc chelate chromatography. The AAV may berecovered from the columns as follows: hydroxyapatite (at 100-135 mMphosphate, pH 6.4); DEAE-ion exchange (at 200 mM salt in a phosphatebuffer, pH 7.5); Cellufine® sulfate (at 425 mM salt inphosphate-buffered saline, pH 7.5) and in the flow-through from the zincchelate column (Hepes buffer, pH 7.5).

A particularly preferred embodiment for purification of AAV includes thefollowing steps: pressure lysis of AAV-infected 293 cells also infectedwith adenovirus in the presence of Tween-80 and trypsin, and recovery ofvirus in the lysate; clarification of lysate via filtration through a0.45 μm or 0.8 μm cellulose acetate filter; ceramic hydroxyapatitechromatography (CHA), where bound AAV is eluted from the resin in100-135 mM phosphate, pH 6.4; DEAE ion-exchange chromatography of theCHA eluate using a MemSep® cartridge, where bound AAV is eluted from theresin in 200 mM salt (phosphate buffer, pH 7.5); Cellufine® sulfatechromatography of the DEAE-eluate, where bound AAV is eluted from theresin in 425 mM salt (phosphate-buffered saline, pH 7.5); and,optionally, zinc chelate chromatography where AAV is recovered in theflow through fraction (Hepes buffer, pH 7.5).

AAV may also be separated from adenovirus using Superdex® 200 resin(Pharmacia, Piscataway, N.J.), which separates AAV from low molecularweight contaminants, and where AAV is recovered in the void volume.

The methods described here permit retrieval of purified AAV particles athigh concentration in aqueous media without centrifugal pelleting.Similarly the methods are suited to the preparation of milligramquantities of virus without the use of density centrifugation.

The use of other columns is also within the scope of the invention whichis directed to the use of column chromatography in large scalepurification of AAV.

In order to assess the integrity of a purification protocol, one skilledin the art can use any number of assays to determine whether AAV virusis recovered and whether cellular proteins and contaminating helpervirus (such as adenovirus) have been removed. AAV recovery andpurification can be monitored by determining the levels of

AAV DNA or AAV proteins in recovered fractions from the variouschromatography steps, or from the titer of infectious virus.

The level of AAV DNA may be determined using a slot blot apparatus whichdetects immobilized DNA using an AAV-specific probe. The number of viralparticles can be determined with the use of a standard curve generatedfrom samples of known particle number. Where recombinant AAV contains amarker gene, such as β-galactosidase, the amount of recovered virus canbe determined by an appropriate assay for the marker gene product (e.g.,X-gel) or by an assay that detects DNA copies of the gene (e.g., PCR).

Alternatively, the presence of virus may be determined from the level ofAAV protein contained in recovered fractions. Viral proteins may beassayed by Western blotting, immunoprecipitation, Coomassie-stainedSDS-PAGE gels, or any other methods for protein characterization andquantitation known to those skilled in the art. When an SDS-PAGE gel isstained with Coomassie Blue, the presence of other non-AAV proteins maybe determined as an index of the concentration of the AAV fraction.

Purity of the isolated virus fraction is determined by SDS-PAGE analysisof proteins in the fraction, followed by Coomassie staining anddensitometry. With respect to the AAV viral proteins, VP3 usuallyaccounts for about 80% of the viral protein, while VP1 and VP2 togetheraccount for about 20% of total viral protein. Purity is assessed by theabsence of heterologous proteins in assayed sample.

The purification methods of the invention may be applied to naturallyoccurring or recombinant viruses.

The practice of the invention employs conventional techniques ofmolecular biology, protein analysis and microbiology which are withinthe skill of the art. Such techniques are explained fully in, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley& Sons, New York, 1995, which is incorporated herein by reference.

The invention is illustrated with reference to the following examples.

EXAMPLE 1 Extraction of Adenovirus from 293 Cells

A. Extraction of adenovirus from cells

The human embryonal cell line (293) was used to propagate adenovirus.Virus-infected cells were incubated until the cell monolayer exhibitedextensive cytopathic effects (CPE). Usual infection time was 48-60hours. The cells were harvested into PBS (phosphate buffered saline) andcollected by centrifugation at 1000×g. Cell pellets were frozen at -80°C. for further use or were resuspended in PBS containing 0.1% Tween-80,10% glycerol, 2 mM MgCl₂ and 50 μM ZnCl₂. Following resuspension thecells were lysed using a Microfluidiser (Model HC5000, Microfluidics,Newton, Mass.) at 1000 psi and the lysate was incubated with benzonase(2500 units benzonase®/10⁸ cells) for 1 hour at room temperature. Toremove cellular debris, the lysate was clarified by passing it throughglass wool (without vacuum) or by vacuum filtration through 3.0 μm glassfiber filters (MicroFiltration Systems #C300A090C, Sierra Court-Dublin,Calif.). This was followed by filtration using a 0.8 μm celluloseacetate filter (MicroFiltration Systems #CD80A090C, Sierra Court-Dublin,Calif.). Following clarification the lysate was either directlychromatographed or subjected to a further filtration step using aMinitan ultrafiltration system (Millipore, #XX42MT060, Bedford, Mass.)prior to chromatography.

B. Ultrafiltration

Lysate from 293 cells infected with adenovirus (prepared as discussedabove) was passed through a Minitan Ultrafiltration System (Millipore#XX42MT060, Bedford, Mass.) at flow rates varying from 300-400 ml/min inthe following buffer: phosphate buffered saline (PBS), 0.05% Tween-80,10% glycerol, 50 μM ZnCl₂. To measure recovery of infectious unitsfollowing ultrafiltration the retentate was assayed for adenovirusinfectivity using the virus titer assay while recovery of protein in theretentate was measured using a BCA assay (Pierce Chemical Co. #23220,Rockford, Ill.).

RESULTS

Table 1 provides a comparison between recovery of active adenovirususing both Microfluidiser pressure lysis and freeze-thawing as methodsof lysis for adenovirus-infected cells. Using the microfluidiser anddetergent-containing buffers, there was a 96% recovery of active viruswith pressure lysis compared to lysis by freeze thawing. Thus, theMicrofluidiser provides an alternative effective lysis procedure, whichhas the advantage of allowing larger volumes of cells to be processed atone time. Also, methods to scale up cell culture conditions, includinggrowing cells on microcarriers, are possible since the microfluidisercan effectively lyse cells attached to microcarriers. Lysis of the cellsoccurred in the presence of the nuclease Benzonase®, which degrades hostcell, nonencapsulated or incomplete adenoviral nucleic acids.

Following lysis of the cells, the resulting lysate was clarified toremove cellular debris by filtration through glass wool or alternativelyby using vacuum filtration through a 3.0 μm glass fiber filter(MicroFiltration Systems #300A090C, Sierra Court-Dublin, Calif.). Afurther filtration step using a 0.8 μm cellulose acetate filter(MicroFiltration Systems) was then carried out. Typically, 84% of activeadenovirus was recovered in the final clarified lysate, while only 43%of total cell lysate protein was recovered.

Ultrafiltration was then used to further purify the clarified celllysate prior to column chromatography. The molecular size of adenovirusis 150×10⁶ daltons, while the molecular size of the majority of hostcell contaminating proteins is expected to be lower. A Minitan®ultrafiltration system from Millipore (molecular weight cut-off membraneof 300 kDa) was used. Table 2 shows that the maximum recovery ofinfectious adenovirus units was achieved when the flow rate through themembrane was 200 ml/min and the buffer contained glycerol and trypsin.Under these conditions 100% of adenovirus activity was achieved while55% of host cell proteins was removed. Spinner cultures of 293 cells(grown on microcarriers) and infected with adenovirus were used in thesestudies. Therefore, effective cell lysis and release of activeadenovirus from 293 cells grown on microcarriers is possible using theMicrofluidiser® pressure cell.

                  TABLE 1                                                         ______________________________________                                        Comparison of different methods for lysis                                       of 293 cells infected with adenovirus                                                     I.U. Total                                                                             % Activity recovered                                   ______________________________________                                        Freeze Thaw (3X)                                                                            4.3 × 10.sup.11                                                                  100                                                      Microfluidiser 4.1 × 10.sup.11  96                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Ultrafiltration of Adenoviral Cell lysates                                      using a Minitan System (Millipore)                                            Flow Rate (ml/min) 400      300     200                                     ______________________________________                                        I.U. before ultrafiltration (×10.sup.10)                                                 6.4      33.44     3.2                                         I.U. after ultrafiltration (×10.sup.10) 2.42 11.73 3.2                  % Adenovirus activity recovered 38 70 100                                     % Total protein remaining 70 57.5 52                                        ______________________________________                                    

EXAMPLE 2 Chromatographic Purification of Adenovirus

Column resins were tested for their separation characteristics using aPharmacia FPLC.

METHODS

1. DEAE Chromatography

A. Adenovirus

A DEAE MemSep® 1010 HP (Millipore) column (5 ml) was equilibrated withphosphate buffered saline (PBS) (1.5 mM KH₂ PO₄, 150 mM NaCl, 5 mM Na₂HPO₄ pH 7.5) containing 10% glycerol, 0.05% Tween-80, and 50 μM ZnCl₂.Clarified lysate from 293 cells infected with adenovirus as prepared inExample 1 was applied at a flow rate of 5 ml/min to the pre-equilibratedcolumn (in PBS, 10% glycerol, 0.05% Tween-80, 2 mM MgCl₂, 50 μM ZnCl₂).The column was washed with 10 mM Na₂ HPO₄, 100 mM NaCl, 100 mM KCl, anda linear gradient (100 mM-1 M) of KCl and NaCl in 10 mM Na₂ HPO₄ pH 7.5,10% glycerol, 0.05% Tween-80 and 50 μM ZnCl₂ was applied to the resin ata flow rate of 5 ml/min. Bound proteins were eluted from the resin andcollected in 5 ml fractions. Each fraction was monitored for a)adenoviral DNA and b) adenoviral proteins (as described below).Fractions which were positive for both adenoviral DNA and protein wereassayed further for activity using the virus titre assay.

B. AAV

Cell lysates from 293 cells infected with adeno-associated virus AAVwere also chromatographed using DEAE MemSep® chromatography as above.However, the buffer used to lyse the cells and equilibrate the columnwas 10 mM sodium phosphate, pH 7.5, containing 50 mM NaCl and 1% NP-40.Bound proteins were eluted from the resin using a salt gradient asdescribed above for adenovirus purification. Fractions collected fromthe resin were assayed for AAV DNA using a slot blot assay and AAVproteins by immunoblotting using an antibody (Catalog 03-65158, ofAmerican Research Products, Belmont, MA) against the three capsidproteins of AAV, VP1, VP2 and VP3.

2. Gel Filtration Chromatography

A. Adenovirus

Gel filtration chromatography was then performed using a Superdex® 200HR 26/60 column (Pharmacia) equilibrated with PBS, 10% glycerol, 2 mMMgCl₂,50 μM ZnCl₂, and 0.05% Tween 80. Fractions eluted from the DEAEresin which showed the presence of both adenoviral proteins and DNA werepooled and concentrated using a stir cell (Amicon) to a volume of 15 ml.The sample was applied to the Superdex® resin at a flow rate of 1 ml/minand 1.5 ml fractions were collected during elution. Fractions wereassayed for adenoviral DNA and proteins as described below.

B. AAV

Adenovirus purified by the cesium chloride method (described below) wasapplied directly to the Superdex® resin following the final cesiumchloride density centrifugation. This was to reduce the concentration ofcesium chloride in the sample which normally was removed by dialysis.

In some experiments gel filtration chromatography of whole cell lysatesof AAV was performed using a Superdex® 200 HR 26/60 prior tochromatography on a DEAE column.

Additional polyermic materials useful according to this aspect of theinvention include cross-linked cellulose polymers such as SulfateSpherilose (ISCO).

Results

FIG. 2 shows a typical elution profile of adenovirus from DEAE MemSep®resin following chromatography of a 293 cell lysate containingadenovirus. All of the adenovirus bound to the DEAE resin and was elutedwith a salt gradient applied to the resin (represented by the slopingline). Adenovirus eluted from the resin between 500-700 mM NaCl asindicated on the elution profile. This peak contained less than 10% ofthe total protein in the initial whole cell lysate, while typically60-100% of the adenovirus activity was recovered. Further purificationof this eluted fraction was achieved by gel filtration chromatographyusing a Superdex® 200 resin. Fractions from the DEAE column which hadthe highest virus titers were pooled, concentrated using an Amicon stircell and applied to the Superdex® resin. Adenovirus eluted in the voidvolume of the resin (FIG. 3). Approximately 50-70% of the adenovirusactivity was recovered in this fraction. Protein estimation (BCA)(Pierce Chemical Co.) on all of the fractions eluted from the columnindicated that the gel filtration step removed approximately 70% ofcontaminating cellular proteins. FIG. 4 shows an SDS-PAGE analysis ofadenovirus purified by a combination of DEAE and gel filtration columnchromatography compared to adenovirus purified by a prior art cesiumchloride method. There were some additional protein bands present in theadenovirus purified by column chromatography. To achieve furtherpurification of the adenovirus other resins were evaluated for theirability to remove these contaminating proteins.

EXAMPLE 3 Hydrophobic Chromatography

Methods

Four different types of hydrophobic resins were tested for their abilityto remove contaminants from the adenovirus preparations of Example 2:BioRad Macroprep® columns (butyl and methyl) and Tosohaus® 650 M, 65 μm(phenyl and ether). Adenovirus was applied to each hydrophobic resin in10 mM sodium phosphate pH 7.5 buffer containing 2 M NaCl. Bound proteinswere eluted from the resin using 0.15 mM KH₂ PO₄, 15 mM NaCl, 0.5 mM Na₂HPO₄ pH 7.5.

Results

SDS-PAGE analysis of the flow-through and eluted fractions showed thatthere was little separation of the adenovirus from other cellularcomponents using these resins.

EXAMPLE 4 Cation Exchange Resins

Methods

CM and SP MemSep® Cartridges (Millipore, Bedford, Mass.), Fractogel® SO₃(a tentacle ion-exchange resin, EM Sciences), and BioRad Macroprep S®were separately equilibrated in 10 mM sodium phosphate buffer pH 7.5,containing 25 mM NaCl, 2 mM MgCl₂, 10% glycerol and 0.05% Tween-80. Celllysates from 293 cells infected with adenovirus were applied to each ofthe resins in the same buffer containing 0.25% Tween-80. The column waswashed with 10 mM Na₂ HPO₄, 100 mM NaCl, 100 mM KCl, and bound proteinswere eluted from the resin using a linear gradient (100 mM-1 M) of KCland NaCl in 10 mM Na₂ HPO₄ pH 7.5, 10% glycerol, 0.05% Tween-80 and 50μM ZnCl₂. The results of the Macroprep S® chromatography are shown inTable 5.

EXAMPLE 5 Cellufine® Sulfate Resin (Amicon)

Methods

For all experiments with the Cellufine® sulfate resins (Amicon, Beverly,Mass.), cell lysate from 293 cells infected with adenovirus was appliedto the resin in a solution of 25 mM NaCl and 10 mM sodium phosphate, pH7.5, containing also 10% glycerol (w/v), 0.05% Tween-80, 2 mM MgCl₂, 50μM ZnCl₂. Bound proteins were eluted from the resin using a linear saltgradient (100 mM-1 M) of NaCl and KCl in 10 MM Na₂ HPO₄ 12 pH 7.5, 10%glycerol, 0.05% Tween-80 and 50 μM ZnCl₂. Both the flow through andeluted fractions were assayed for adenoviral DNA and immunoblotted usingan anti-adenoviral antibody as described below.

Results

The resin which gave the most significant purification, Cellufine®sulfate, did not however lead to a purified product in which most of theadenovirus was present in an active form. Cellufine® sulfate comprises acellulose matrix with sulfonate groups esterified at the number -6carbon of the repeating glucose subunits (P. F. O'Neil et al.Biotechnology, 11, 1993, pp.173-178). Binding of proteins to this resinis thought to occur through the polysaccharide moieties thereof. Becauseadenovirus is a non-enveloped virus with no surface glycoproteins, itwas thought that it should not bind to this resin while most cellularglycoproteins, present as contaminants, would.

Table 3 shows the results for recovery of activity of adenovirusfollowing chromatography on the Cellufine® sulfate. Adenoviral proteinand DNA were recovered in the flow through volume, as predicted, butless than about 10% of adenoviral activity was recovered in thisfraction. Inactivation of the virus during chromatography on Cellufine®sulfate may have been a result of adverse interaction/partitioninginvolving the pores of the beads of the resin, which have a meandiameter of about 80 nm.

EXAMPLE 6 Heparin Resins

Methods

Each of the following heparin resins were assessed for their ability topurify adenovirus: Heparin Sepharose® CL6B (Pharmacia), Heparin Agarose(4% cross linkage, Sigma), HiTrap® Heparin (Pharmacia), HeparinSuperflow Plus® (6% cross linkage, Sterogene), Heparin, ACTI-MOD®cartridge® (American International Chemical Inc.,).

For all experiments with the heparin resins, cell lysate from 293 cellsinfected with adenovirus was applied to the resin in a solution of 25 mMNaCl and 10 mM sodium phosphate, pH 7.5, containing also 10% glycerol(w/v), 0.05% Tween-80, 2 mM MgCl₂, 50 μM ZnCl₂. Bound proteins wereeluted from the resin using a linear salt gradient (100 mM-1 M) of NaCland KCl in 10 mM Na₂ HPO₄ pH 7.5, 10% glycerol, 0.05% Tween-80 and 50 μMZnCl₂. Both the flow through and eluted fractions were assayed foradenoviral DNA and immunoblotted using an anti-adenoviral antibody asdescribed below.

Results

In order to further understand the causes for the disappointingperformance of Cellufine® sulfate, the performance of heparinatedpolymers (resins) was also examined. Heparin, like Cellufine® sulfate,is a sulfonated polysaccharide and would be predicted to have certainbinding characteristics in common. Unlike Cellufine® sulfate, however,it is commercially available in one or more forms cross-linked to avariety of different beaded resins of various pore size. Table 3 showsthe results of screening various heparin-linked resins. All of theresins tested bound >40% of the cellular proteins (as determined by BCA)that contaminated the virus samples, but the only resin which gave 100%recovery of active virus was the Sterogene® heparin agarose, a 6%cross-linked agarose. Generally speaking, a 6% agarose matrix with anexclusion limit of about 6 million daltons would have smaller poresthan, for example, an agarose gel with 4% cross-linkage and an exclusionlimit of about 20 million daltons. It is possible that, because of itsaverage pore size, the 6% cross-linked agarose excluded the adenoviruscompletely during chromatography. As a result, active adenovirus wasrecovered in the flow-through fraction.

                  TABLE 3                                                         ______________________________________                                        Recovery of Active Adenovirus following                                         chromatography with Cellufine ® Sulfate and Heparin                       Resins                                                                                       I.U. before I.U. after                                                                           %                                            Chromato- Chromato- Activity                                                 Resin graphy graphy Recovered                                               ______________________________________                                        Heparin      2.1 × 10.sup.10                                                                     5.7 10.sup.9                                                                           27                                            Sepharose ® CL6B                                                          HiTrap ® Heparin 5.8 × 10.sup.10 3.8 × 10.sup.9 7                                              Heparin Agarose, 5.8 × 10.sup.10                                       9.2 × 10.sup.9 16                       4% X-link                                                                     Heparin 5.1 × 10.sup.9 1.3 × 10.sup.10 100                        Superflow ® Plus                                                          6% x-link agarose                                                             Cellufine ® -- -- 10                                                      Sulfate                                                                     ______________________________________                                    

Lysate from 293 cells infected with adenovirus was chromatographed usinga heparin ACTI-MOD® disc as described above. Adenovirus was recovered inthe flow through fraction and purified further using a combination ofDEAE ion exchange and gel filtration chromatography. SDS-PAGE analysisof the adenovirus fraction from the GF column showed that the purity ofthe adenovirus (purified by column chromatography) was as pure as thecontrol adenovirus purified by CsCl centrifugation (FIG. 5). FIG. 6shows a densitometric analysis of the fractions analyzed in FIG. 5.

EXAMPLE 7 Biorad Ceramic Hydroxyapatite (80 μm pore size)

The BioRad hydroxyapatite column was equilibrated with 10 mM sodiumphosphate pH 7.5 containing 25 mM NaCl,Y 1 mM MgCl₂ and 10% glycerol.Adenovirus or AAV was applied to the resin in the same buffer. Boundproteins were eluted from the resin using an increasing linear saltgradient from 10 to 300 mM of sodium phosphate, all at pH 7.5.

EXAMPLE 8 Partitioning Polymers

A series of partitioning polymers (resins) were screened for theirability to purify active adenovirus (Table 4). It was found that themajority of the polymers tested gave significant purification, but useof only a few led to recovery of purified adenovirus in an active form.In general, the membrane-based cartridge polymers (resins), such as theMemSep® cartridge from Millipore or the ACTI-MOD® cartridge (AmericanChemical International) gave a better recovery of active virus. Thesuperior performance of these products is believed attributable to theopen macroporous structure of the membrane matrix in these cartridges[in these preferred examples, DEAE groups are covalently linked to thepolymer matrix cellulose in the case of MemSep®, or to silicate in thecase of ACTI-MOD®]. This type of macroporous structure [having openings(pores) of about 12,000 A°, or 1.2 μm, in width] allows rapid passage ofthe adenovirus virions which have diameters of about 140 nm (includingfibre).

Table 5 is a summary chart of various chromatographic methods foradenovirus purification.

                  TABLE 4                                                         ______________________________________                                        Recovery of Active Adenovirus from Different                                    Types of Resins                                                               Type of Resin     Pore Size  Activity Recovered                             ______________________________________                                        Membrane Based  1 μm    100%                                                 (Memsep ® or ACTI-MOD ®)                                              Macroporous (BioRad) 0.08-0.1 μm   10%                                     Tentacle (EM Science) ND <10%                                                 4% Agarose ND <20%                                                            6% Agarose ND 100%                                                          ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Results of Resin Screen                                                                                     CLEAN-                                            RESIN TYPE UP ACTIVITY                                                      ______________________________________                                        Sperilose Sulfate                                                                         cross-linked cellulose                                                                      good       62%                                        (ISCO)                                                                        Heparin Superflow 6% cross-linked good   80%                                  Plus                                                                          POROS PI 6000-8000 A thru- fair    5%                                          pores                                                                        Fractogel DEAE (EM "tentacles" rather good   35%                              Sciences) than pores                                                          DEAE MemSep ® Membrane based good   80%                                   SartoBind DEAE membrane based good   30%                                      (Sartorius)                                                                   PolyFlo ® (Puresyn) non-porous excellent    4%*                           Heparin Agarose 4% cross-linked good   16%                                    (Sigma) agarose                                                               HiTrap Heparin cross-linked agarose good    7%                                (Pharmacia)                                                                   Heparin CL6B cross-linked agarose good   16%                                  Cellufine ® MacroPorous excellent <10%                                    Sulfate (Amicon)                                                              Hydroxyapatite calcium phosphate good <10%                                    (BioRad)                                                                      MacroPrep S (BioRad) macroporous    40%**                                   ______________________________________                                         *Purification of adenovirus on the PolyFlo resin involves low salt and        organic elution.                                                              **Batch Process                                                          

EXAMPLE 9 Purification of Adeno-Associated Virus (AAV)

One of the main problems associated with using AAV as a vector in genetherapy is production of sufficient quantities of the virus. CurrentlyAAV is purified by density gradient ultracentrifugation techniques,which generally results in very low yields (0.3-5%) of active virus.However density gradient ultracentrifugation is very effective inseparating AAV from adenovirus, which is used as a helper virus inpropagating AAV in 293 cells. The present invention provides combiningan improved method for extraction of AAV from infected cells with columnchromatography steps to increase the yield of AAV.

Improved extraction of AAV from infected 293 cells was achieved bypressure lysis of the cell in the presence of detergent (Tween-80).Following clarification of the lysate as provided above in Example 1,AAV was separated from other cellular proteins by gel filtrationchromatography. FIG. 7a shows the elution profile from a Superdex® 200resin (Pharmacia) following chromatography of AAV infected 293 celllysate. The void volume peak contains the majority of the AAV asdetected by slot blot analysis and immunoblotting of this fraction.

Further purification of this peak was achieved by ion-exchangechromatography using a DEAE-MemSep® cartridge. The DEAE column was veryeffective in separating AAV from adenovirus. Under the conditions used(10 mM sodium phosphate pH 7.5 containing 25 mM NaCl, 10% glycerol and0.05% Tween-80), both AAV and adenovirus bound to the DEAE-resin. When alinear salt (KCl and NaCl) gradient was applied to the resin, AAV elutedat 200 mM salt (FIG. 7, panel B), while the adenovirus remained moretightly bound to the resin and was eluted later in the gradient at500-700 mM NaCl (FIG. 7, panel B). Therefore, AAV and adenovirus can beeffectively separated from one another using DEAE ion-exchangechromatography. SDS-PAGE analysis of two DEAE fractions containing AAVis shown in FIG. 8. The activity of AAV in the DEAE fraction #7 was6.5×10⁷ i.u./ml or a total of 3.8×10⁸ i.u. The activity of AAV in theDEAE pool fraction was 1.38×10⁷ i.u./ml or a total of 2.76×10⁸ i.u.Collectively, these fractions provide recovery of approximately 100%, ofthe AAV infectious units applied to the DEAE resin.

EXAMPLE 10 Extraction of AAV from 293 Cells

The human embryonal cell line (293) was also used to propagate the AAV.Virally infected cells were incubated until the cell monolayer exhibitedextensive cytopathic effects (CPE). The cells were harvested andcollected by centrifugation at 1000×g. Cell pellets were frozen at 80°C. for further use or were resuspended in 10 mM NaPi, 10 mM NaCl, 10%glycerol, 2 mM MgCl₂, pH 6.4.

Following resuspension, the cells were treated with benzonase® for 1hour at room temperature followed by trypsin treatment in the presenceof 1% Tween-80. The cells were then lysed using a Microfluidiser(Microfluidics, Newton, Mass.) at 1000 psi. The resulting lysate wasclarified to remove cellular debris by vacuum filtration through a 3.0μm glass fiber filter (Microfiltration Systems), followed by a furtherfiltration step using a 0.8 μm cellulose acetate filter (MicrofiltrationSystems) or filtered through a 0.45 μm Millex® HV (Millipore) filterunit before chromatography.

EXAMPLE 11 Chromatographic Purification of AAV

Various chromatography resins were tested for effective AAV purificationcharacteristics using a Pharmacia FPLC. The following series ofchromatography steps were found particularly useful.

a) BioRad Ceramic Hydroxyapatite (80 μm pore size)

Cell lysates from 293 cells infected with AAV (in the presence ofadenovirus) (Example 10) were chromatographed on a BioRad ahydroxyapatite column, which was pre-equilibrated with 10 mM Na₂ HPO₄ pH6.4, containing 10 mM NaCl and 10% glycerol. AAV was applied to theresin in the same buffer. Bound proteins were eluted from the resinusing an increasing gradient from 10 to 400 mM sodium phosphate, at pH6.4. Fractions collected from the resin were assayed for AAV DNA using aslot blot assay and AAV proteins by immunoblotting using an antibody(Catalog 03-65158, from American Research Products, Belmont, Mass.)against the three capsid proteins of AAV-VPI, VP2 and VP3. Fractionseluted from the resin were also analyzed for adenoviral contaminatingproteins by immunoblotting using an anti-adenoviral antibody.

FIG. 9A shows a typical elution profile of a ceramic hydroxyapatite(CHA) resin following chromatography of a 293 cell lysate containing AAVand adenovirus. All of the AAV and adenovirus bound to the CHA resin andwas eluted when a phosphate gradient was applied to the resin. AAVeluted from the resin at 125 mM phosphate as indicated on the elutionprofile. This peak contained less than 20% of the total protein in theinitial whole cell lysate while typically 80% of the AAV activity wasrecovered. The eluted AAV peak also contained some contaminatingadenoviral proteins as measured by immunoblotting and by titre analysis(Table 5).

b) DEAE Chromatography (anion exchange)

In order to separate AAV from the adenovirus, an ion exchangechromatography using a DEAE-MemSep® column was used. A DEAE MemSep® 1010HP (Millipore) column (5ml) was equilibrated with 10 mM phosphate buffercontaining 50 mM NaCl, 10% glycerol, pH 7.5. AAV-containing fractionseluted from the hydroxyapatite column (above) were pooled and dialyzedinto the same buffer used for equilibration of the DEAE resin. A linearsalt gradient (50 mM-2 M) of KCl and NaCl in 10 mM Na₂ HPO₄ pH 7.5, and10% glycerol was applied to the resin at a flow rate of 5 ml/min. Boundproteins were eluted from the resin and collected in 2.5 ml fractions.AAV eluted at 200 mM salt while adenovirus eluted at 500-700 mM salt.Each fraction was monitored for a) AAV proteins (Coomassie blue stainingand immunoblotting); b) AAV DNA; c) contaminating adenoviral proteins;and (d) infectivity.

Under the conditions used (10 mM sodium phosphate pH 7.5 containing 50mM NaCl, 10% glycerol and 0.05% Tween-80) both AAV and adenovirus boundto the DEAE-resin. When a linear salt gradient was applied to the resin,AAV eluted at 200 mM salt (FIG. 9B) while the adenovirus remained moretightly bound to the resin and was eluted later in the salt gradient at500-700 mM NaCl (FIG. 9B). Therefore AAV and adenovirus can beeffectively separated using anion exchange (DEAE) chromatography. Therecovery of activity of AAV from the DEAE-MemSep® was 75% (Table 5).SDS-PAGE analysis of the pooled-AAV containing fraction from the DEAEresin (FIG. 10, lane 3) showed that there were still some contaminatingproteins present so this fraction was purified further using aCellufine® sulfate resin. Lanes 1-4 represent equal percentages of eachfraction (0.5%) and show the recovery of AAV proteins throughout thepurification. Lane 5 represents a larger percentage of the final AAVcontaining fraction and gives a more intense staining of the AAVproteins VP1, VP2 and VP3. Fractions which were positive for both AAVDNA and protein were pooled and chromatographed further using aCellufine® sulfate resin (below).

c) Cellufine® sulfate resin (Amicon)

Cellufine® sulfate resin was equilibrated with PBS containing 10%glycerol. Fractions eluted from the DEAE resin which contained AAVproteins and DNA were pooled and applied to the resin at a flow rate of1 ml/min. The resin was washed with 250 mM NaCl and a linear saltgradient of 0.25-1 M NaCl in PBS/glycerol was applied. The materialeluted from the resin using this salt gradient and the flow throughfraction were both analyzed for a) AAV proteins (immunoblotting); (b)AAV infectivity (titre analysis); and c) adenovirus proteins(immunoblotting) and adenovirus infectivity (titre analysis). Under thebuffer conditions used, AAV bound to the resin and was eluted at 475 mMsalt (FIG. 9C). Adenoviral protein and DNA were recovered in the flowthrough fraction.

Table 5 shows the recovery of AAV activity from several purificationruns. FIG. 10 shows Coomassie Blue-stained SDS-PAGE results of thepurification from each column. The three column purification proceduredescribed above provided an AAV yield of 48% and a purity of >90% pure(FIG. 11). FIG. 12 is a Coomassie stained gel comparing AAV purifiedusing the CsCl gradient method and AAV purified using the columnpurification procedure of the present invention. This gel shows that theAAV purified using both methods is of comparable purity. In addition,the AAV purified by the above procedure was shown to be free of Repproteins (FIG. 13). FIG. 13 shows an immunoblot (using an anti-Repantibody) of the column-purified AAV along with known Rep standards.Approximately 71% of the AAV activity is recovered in the Cellufine®sulfate eluate while less than 1% of the adenovirus activity isrecovered in the same fraction.

Cellufine® sulfate thus has two main uses: a) it reduces the level ofcontaminating adenovirus in AAV preparations and b) it concentrates theAAV containing fraction. However, despite the fact that the level ofcontaminating adenovirus is reduced following Cellufine® sulfatechromatography there still remains some low level adenovirus activity(6.73×10³ IU/ml).

d) Zinc Chelate Chromatography

In order to completely remove all of the contaminating adenovirus afurther chromatography step using zinc chelate chromatography wasemployed. The interaction of virions with metals has been inferred fromstudies of viruses and bacteriophages. Previous studies from theinventors' laboratory showed that adenovirus can adsorb to a zinc metalaffinity column.

The final fraction of purified AAV recovered from the Cellufine® sulfateresin was analyzed by SDS-PAGE, followed by immunoblotting using ananti-adenovirus antibody. This was to determine the level ofcontamination of adenovirus in the final AAV containing fraction.Immunoblotting showed that there was no detectable adenoviral proteins,while titre analysis showed that there was some adenoviral activity inthis fraction even though it only accounted for 1% of the total activity(Table 6).

The immobilized zinc column was prepared for metal charging by washingthe column sequentially with one volume of 100 mM EDTA and one volume of0.2M NaOH. The matrix was charged with zinc by washing with 100 mM ZnCl₂in water acidified with glacial acetic acid. The column was then washedwith water and equilibrated with 50 mM Hepes pH 7.5, containing 450 mMNaCl, 2 mM MgCl₂ and 0.05% Tween-80. The AAV-containing fraction elutedfrom the Cellufine® sulfate resin was applied to the zinc chelate resin.After loading, the column was washed with a ten column volume lineargradient from 50 mM Hepes, pH 7.5, containing 450 mM NaCl, 2 mM MgCl₂,10% glycerol and 0.05% Tween-80 to 50 mM Hepes containing 150 mM NaCl, 2mM MgCl₂, 10% glycerol and 0.05% Tween-80. Elution was performed with alinear (0-500 mM) glycine gradient in 150 mM NaCl, 50 mM Hepes, pH 7.5,2 mM MgCl₂ over ten column volumes.

The AAV containing fraction eluted from the Cellufine® sulfate resin wasapplied to the zinc chelate resin in 450 mM NaCl. The flow throughfraction was collected and bound proteins were eluted using a glycinegradient. SDS-PAGE analysis of the flow through fraction showed that allof the AAV was recovered in the flow through while immunoblots using ananti-adenovirus antibody showed that the adenovirus had bound to thezinc chelate resin and was eluted in the presence of an increasinggradient of glycine. Initial experiments using zinc chelatechromatography indicates that it can be a useful resin for theseparation of AAV and adenovirus.

The purification procedure yielded AAV which was greater than 70% purewith an overall yield of 30%-40%.

                  TABLE 6                                                         ______________________________________                                        Summary of AAV Chromatographic Purification                                   ______________________________________                                        Column Performance: Total Protein                                                                      % Protein % Protein                                    Sample Total Protein Remaining.sup.1 (cumulative).sup.2                     ______________________________________                                          HA Load 180 mg  110 100                                                       HA Eluate 20 mg 11 11                                                         DEAE Eluate  6 mg 30 3                                                        CS Eluate 0.5 mg  8 0.4                                                     ______________________________________                                        Column Performance: Recovery of AAV Activity                                    Sample     #1     #2    #3    #4    #5    average                           ______________________________________                                          HA Load                                                                       HA Eluate 84% 86% 60% 100%  100%  88%                                         DEAE Eluate 75% 100%  100%  56% 18% 70%                                       CS Eluate 71% 24% 30% 22% 38% 48%                                             Zinc FT  9% 9%   20%                                                        ______________________________________                                        Column Performance: Adenovirus Activity Removed                                 Sample     #1            #2    average                                      ______________________________________                                          HA Load                                                                       HA Eluate 90% 0*  90%                                                         DEAE Eluate 95% 70% 80%                                                       CS Eluate >99%   >99%   >99%                                                  Zinc FT  50% 50%                                                            total >99.99% of contaminating adenovirus activity removed                    ______________________________________                                          Average Activity Recovered during Purification Runs (n = 2):                                                 DEAE    cs                                      HA Load HA Eluate Eluate Eluate                                            ______________________________________                                          total mgs 236 13 5 0.6                                                        AAV IU/ml 3.58 E + 07 1.31 E + 08  5.4 E + 08 2.42 E + 08                     Total AAV IUs 3.92 E + 09 7.24 E + 09 2.47 E + 09 1.11 E + 09                 AAV IUs/ug 1.66 E + 04 5.57 E + 05 4.94 E + 05 1.85 E + 06                  ______________________________________                                         .sup.1 Individual column performance                                          .sup.2 Overall performance                                                    *not included in the average                                             

The average adenovirus activity remaining (calculated from 2 runs) after3 columns=4.8E+04 (+/-4E+04) Adenovirus activity represents 0.03%+/-0.015% of the total AAV activity

EXAMPLE 12 Density Gradient Purification of Virus

Standard recombinant adenovirus or AAV virus was prepared by a threestep centrifugation procedure. Infected cells were lysed by three cyclesof freeze-thaw in the presence of benzonase®. Lysate was centrifuged ina table top centrifuge for 15 min at 3500 rpm at 4° C. The pellet wasdiscarded and the supernatant was layered onto a 1.27 g/cm³ CsCl and 1.4g/cm³ CsCl discontinuous step gradient and centrifuged at 26,000 rpm for1.5 hours. The virus band was collected and mixed with 1.34 g/ml CsCland centrifuged for at least two hours at 60,000 rpm. The viral bandfrom this first equilibrium gradient was collected mixed with 1.34 g/mlCsCl and recentrifuged at 30,000 rpm overnight. The final virus poolfrom this step was dialyzed extensively against phosphate bufferedsaline (PBS) supplemented with 1% sucrose. Alternatively the CsCl wasremoved by gel filtration on a Superdex® resin (Pharmacia) as describedabove.

EXAMPLE 13 Detection of Adenoviral DNA Using Agarose Gels

Column fractions were first treated with 0.1% SDS for 15 minutes thendigested with Pronase (Sigma) for 1 hour at room temperature. Afterdigestion was complete, the samples were extracted twice with one volumeof phenol: CHCL₃ :isoamyl alcohol, then precipitated with two volumes ofice-cold 95% ethanol for 20 minutes at -20° C. The precipitate waspelleted at 13,000×g for 20 min at 4° C. Samples were resuspended in TEbuffer (Tris, EDTA). Restriction enzyme digestion of the adenoviral DNAwas performed and the digest was analyzed for diagnostic adenoviralfragments on a 0.8% agarose gel at 120 V for 4 hours or overnight at 35V.

EXAMPLE 14 Slot Blot Analysis of Column Fractions for Detection of AAVDNA

Column fractions were assayed for AAV DNA by slot blot analysis (AAV DNAwas present in a vector construct provided by Dr. N. Muzyczka,University of Florida, Miami, containing also lacZ as reporter gene, seeFIG. 14). Samples were incubated at 56° C. for 20 minutes to inactivatethe adenovirus followed by treatment with DNaseI at 37° C. for 15 min todegrade nonvirion DNA. DNaseI was then inactivated by heat treatment at68° C. for 10 minutes. Following Proteinase K treatment of the samples,the DNA was extracted using phenol/chloroform and precipitated with 3 MNaOAc. DNA was applied to a Gene Screen Plus membrane and following aprehybridization and hybridization step the membrane was probed with aP32 random label CMV β-gal Pvu II fragment. The number of particles ofAAV in the sample was calculated using a pTRlacZ DNA standard curve.

EXAMPLE 15 Virus Protein Detection

One dimensional SDS-PAGE was performed using either a 4-20% or 10-20%gradient (Daiichi) gels. Proteins in the gel were detected usingCoomassie blue. For immunoblotting, PVDF membranes (Novex) wereprewetted with methanol and soaked in 10 mM CAPS, pH 11, containing 10%methanol. Gels were equilibrated in this transfer buffer for 10 minutesand then blotted at 30 V for 1 hour in a Novex Blot Module. Aftertransfer membranes were blocked with 1% dried milk in TBS (20 mMTris-HCl, pH 7.5, containing 150 mM NaCl) for one hour. After blocking,the membranes were probed with anti-adenovirus antibody (LeeBiomolecular) or anti-VP1, VP2, VP3 (AAV) antibody in 20 MM Tris-HCl,150 mM NaCl, pH 7.5, and 0.05% Tween 20 (TBST) containing 0.1% BSA for 2hours. The membranes were incubated with horse radish peroxidase labeledanti-mouse IgG for 20 minutes and the immunoreactive bands visualized bychemiluminescence using the BM Chemiluminescent Western BlottingDetection System (Boehringer Mannheim).

What is claimed is:
 1. An improved method for purification ofadeno-associated virus (AAV) from a composition containing both AAV andadenovirus, wherein the improvement comprises inclusion of a step forinactivation and removal of adenovirus, said step comprising mixing thecomposition with a chromatographic matrix material having pores whichare approximately the size of adenovirus, such that adenovirus isinactivated, said chromatographic matrix material being selected fromthe group consisting of:a. "macroporous" resins, such as macroporousDEAE resins and the BioRad macroporous series (Biorad, Melville, N.Y.);b. heparinized polymers, such as Heparin Agarose (4% cross-linked)(Sigma Chemical, St. Louis, Mo.); c. virus-specific antibodies; such asanti-rep antibodies; d. sulfated resins, such as Sterogene-S (SulfatedHi Flow) (Sterogene, Carlsbad, Calif.), Spherilose-S (Isco, Lincoln,Nebr.), and Cellufine® sulfate (Amicon, Beverly, Mass.); e.hydroxyapatite resins, such as ceramic hydroxyapatite resins from Biorad(Melville, N.Y.); f. a series of column separations including the use ofceramic hydroxyapatite, DEAE ion-exchange, Cellufine® sulfate, and,optionally, zinc chelate chromatography.
 2. The method of claim 1,wherein the chromatographic matrix material comprises a macroporous DEAEresin.
 3. The method of claim 1, wherein the chromatographic matrixmaterial comprises a macroporous hydroxyapatite.
 4. The method of claim1, wherein the chromatographic matrix material comprises a cellulose orsilica membrane resin in conjunction with a macroporous resin.